• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.171-178
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• 1999

The follicular fluid (FF) of ovary contains various biological active products which affected on the growth of follicles and the fertilization of oocyte in physiological reproductive process of mammals. This study was designed to determine the effects of human FF on fertilization of oocyte and embryonal development in vitro culture. The FF was prepared as clear without blood contamination by needle aspiration from mature follicles of human at the time of oocytes retrieval for in vitro fertilization (IVF). As the medium for culture in vitro of embryonal cells, human tubal fluid (HTF) supplemented with follicular fluids at concentrations of 10%, 40% and pure FF were used. These effects were compared to control group of cultured embryos in HTF supplemented with 0.4% BSA (bovine serum albumin). For IVF, 64 eggs in control group, 67 eggs in 10% FF, 57 eggs in 40% FF and 64 eggs in pure FF were respectively allocated. And the rates of fertilization were almost similar in all groups as resulting 82.81% in control, 85.07% in 10% FF, 87.71% in 40% FF and 81.25% in pure FF. On the examination for embryonal cleavage from fertilized eggs, the rates of developing to 4 cell stage was similar in all groups, as results 98.11% in control, 98.27% in 10% FF and 98% in 40% FF but 78.84% in pure FF. And the rates of developing to 8-16 cell stage were significantly reduced as 44% in 40% FF and 44.23% in pure FF (p<0.05) compare to 71.69% in control media. As likewise, the rates of developing to morular stage were also significantly reduced to 36% (p<0.05) and 21.15% (p<0.01) respectively in 40% FF and pure FF. And the rates to blastocystic stage of embryo was lowest as 7.69% in pure FF (Table 1). The quality of embryonal cells on cleavage to the 8-16 cell stage was poorer, higher concentrations of FF. The rates of grade 1 in pure FF, as 23.07%, was lowest compare to those of other groups, in which the rates of grade 1 in control, 10% FF and 40% FF were 58.49%, 47.36% and 34% respectively. And on the contrary, the rate of grade 4 in pure FF was highest as 23.07%, while those were 5.66% in control, 8.77% in 10% FF and 20% in 40% FF (Table 2). On the viability of embryos, the rate of embryonal cell death was more rise, at the higher concentrations as well as longer exposure in the follicular fluid. At 48 hours after in vitro culture of embryos, the rate of survival embryos in pure FF was markedly lowered as 44.23%, compare to that of control (p<0.05). But there was not significant difference between the rates of survival embryos in each group beside the pure FF, which the rates were 77.35% in control, 70.17% in 10% FF and 60% in 40% FF respectively. And at 72 hours after in vitro culture, the rates of survival embryos were also significantly dropped to 21.15% in pure and 36% in 40% at concentration of FF compare to 62.26% in control (p<0.05, p<0.01). Finally, the rate of embryonal death at 96 hours after in vitro culture was highest as 82.69% in pure FF among all groups which those were 35.84 in control, 56.14% in 10% FF and 64% in 40% FF respectively (Fig. 1, 2, 3). In conclusion, this study suggests that the FF has no effects, in particular, to the in vitro fertilization of oocytes but exerted a bad effect to the cleavage, quality and viability of the embryonal cells during in vitro culture. However, the FF is harmful on embryonal development at conditions in higher concentration and especially on the embryos after $8{\sim}16$ cell stage.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.47-57
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• 2000

Objective: The effects of cryopreservation with or without coculture on the in vitro development of blastomere-biopsied 8-cell mouse embryos were investigated. This experimental study was originally designed for the setup of a preclinical mouse model for the preimplantation genetic diagnosis (PGD) in human. Methods: Eight-cell embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BL(표현불가)/CBA(표현불가)). Using micromanipulation, one to four blastomeres were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acid Tyrode's solution (ATS). A slow-freezing and rapid-thawing protocol with 1.5M dimethyl sulfoxide (DMSO) and 0.1M sucrose as cryoprotectant was used for the cryopreservation of blastomere- biopsied 8-cell mouse embryos. After thawing, embryos were cultured for 110 hours in Ham's F-10 supplemented with 0.4% bovine serum albumin (BSA). In the coculture group, embryos were cultured for 110 hours on the monolayer of Vero cells in the same medium. The blastocyst formation was recorded, and the embryos developed beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. Results: The survival rate of embryos after cryopreservation was significantly lower in the blastomere-biopsied (7/8, 6/8, 5/8, and 4/8 embryos) groups than in the non-biopsied, zona intact (ZI) group. Without the coculture, the blastocyst formation rate of embryos after cryopreservation was not significantly different among ZI, the zona drilling only (ZD), and the balstomere-biopsied groups, but it was significantly lower than in the non-cryopreserved control group. The mean number of cells in embryos beyond blastocyst stage was significantly higher in the control group ($50.2{\pm}14.0$) than in 6/8 ($26.5{\pm}6.2$), 5/8 ($25.0{\pm}5.5$), and 4/8 ($17.8{\pm}7.8$) groups. With the coculture using Vero cells, the blastocyst formation rate of embryos after cryopreservation was significantly lower in 5/8 and 4/8 groups, compared with the control, 7/8, and 6/8 groups. The mean number of cells in embryos beyond blastocyst stage was also significantly lower in 4/8 group ($25.9{\pm}10.2$), compared with the control ($50.2{\pm}14.0$), 7/8 ($56.0{\pm}22.2$), and 6/8 ($55.3{\pm}25.5$) groups. Conclusion: After cryopreservation, blastomere-biopsied mouse embryos have a significantly impaired developmental competence in vitro, but this detrimental effect might be prevented by the coculture with Vero cells in 8-cell mouse embryos biopsied one or two blastomeres. Biopsy of mouse embryos after ZD with ATS is a safe and highly efficient preclinical model for PGD of human embryos.

• Journal of Life Science
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• v.16 no.4
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• pp.612-617
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• 2006

Phosphoinositide-specific phospholipase $C-{\gamma}\;1\; (PLC-{\gamma}\;1)$ has pivotal roles in cellular signaling by producing second messengers, inositol 1,4,5-trisphosphate $(IP_3)$ and diacylglycerol (DG). Tubulin is a main component of microtubules and mitotic spindle fibers, which are composed of ${\alpha}-$ and ${\beta}-tubulin$ heterodimers in all eukaryotic cells. In humans, six ${\beta}-tubulin$ isotypes have been identified which display a distinct pattern of tissue expression. Previously we found that $PLC-{\gamma}\;1$ and one of four ${\beta}-tubulin$ isotypes including ${\beta}1$, ${\beta}2$, ${\beta}3$ and ${\beta}6$, colocalized in COS-7 cells and cotranslocated to the plasma membrane to activate $PLC-{\gamma}\;1$ upon agonist stimulation. In the present study, we demonstrate that the remaining two, tubulin ${\beta}4$ and ${\beta}5$, also showed a potential to activate $PLC-{\gamma}\;1$. The phosphatidylinositol 4,5-bisphosphate $(PIP_2)$ hydrolyzing activity of $PLC-{\gamma}\;1$ was substantially increased in the presence of purified ${\beta}4$ and ${\beta}5$ tubulin in vitro, whereas the activity was not promoted by bovine serum albumin, suggesting that tubulin ${\beta}4$ and ${\beta}5$ also activate $PLC-{\gamma}\;1$. Taken together, our results suggest that all the ${\beta}-tubulin$ isotype activates $PLC-{\gamma}\;1$ activity to regulate cellular signaling.

• Journal of the Korean Society of Food Science and Nutrition
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• v.33 no.6
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• pp.921-929
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• 2004

Dietary intake of whole grains, vegetable and fruit is known to reduce the degenerative chronic diseases, such as cancer and cardiovascular diseases. Antioxidative and antimutagenic effects of the ethanol extract of Korean Millet, Buckwheat, Sorghum and Job's tears were examined by inhibition against iron-induced linoleate per-oxidation, DPPH (1,l-diphenyl-2-picrylhydrazyl) radical generation and MDA-BSA (malondialdehyde-bovine serum albumin) conjugation, and Ames test using Salmonella. Buckwheat showed the strongest antioxidative effect in three different systems among these four grains, but it showed the lowest antimutagenic effect. Sorghum was the second to Buckwheat in iron-induced linoleate peroxidation inhibition activity and DPPH radical scavenging activity, and showed very good direct-antimutagenic effect in 2-Nitrofluorene treated Salmonella Typhimurium TA98 and indirect-antimutagenic effect in 2-Anthramine treated Salmonella Typhimurium TA98 and TA100 with hepatic S9 mixture. Millet showed the strongest antimutagenic effect in Salmonella Typhimurium TA98 and TA 100 with or without S9. Buckwheat contained the highest total flavonoids and polyphenols, 1.14 mg/g and 3.71 mg/g, respectively. Total flavonoid content in these four grains was negatively correlated with $IC_{50}$/ for DPPH radical scavenging antioxidative effect significantly (r=-0.9924, p=0.0076), but not with antimutagenic effect.